JP2020517257A - Enzyme-based method for extracting value-added products from raw biomass - Google Patents

Abstract

An enzyme-based method for extracting value-added products from oil seeds and grain biomass is described. This method involves an alkaline pretreatment step followed by treatment with a proteolytic enzyme, resulting in increased product yield and solubility. The resulting products can be soluble proteins/peptides and purified dietary fiber. The use of such methods in the manufacture of food, beverage, cosmetic, feed or feed additive products is also described. [Selection diagram]

Description

Cross-reference of related applications

This application claims priority to US Provisional Application No. 62/489,646, filed April 25, 2017, the entire contents of which are incorporated herein by reference.

The present disclosure relates to processing biomass from oil seeds and grains, and more particularly to separating proteins from cellulose-hemicellulose fibers in waste or by-products from oil seeds and other grains.

Generally, the processing of oil seed crops and grain feedstocks into useful food and beverage products produces large amounts of fiber-based waste or by-products. Although these wastes contain valuable protein and fiber components, they are of low concentration and purity and are therefore often disposed of as waste and often costly to remove by the processor. Usually, some oil seed by-products are used as low value animal feed.

For example, the worldwide production of tofu and soy milk from ground soybeans produces millions of tons of solid by-product called okara every year. Okara consists of 75% water. On a dry matter basis, okara contains about 50% dietary fiber, 25% protein, 10% lipids, and other nutrients. However, in its wet form, okara is fermentable and rots after production in a very short time due to its high nutritional value and high water content. Disposal of large quantities of okara has significant environmental and economic problems. At the moment very little okara is used after drying, in the food industry or as animal feed. Most okara is thrown into the fields as fertilizer or incinerated as waste, with losses to both producers and the environment.

Similarly, current methods of processing corn and barley into useful products such as ethanol result in the production of a fiber rich by-product called distillers' dried grain (DDG). Currently, DDG is mostly used in animal feed as a low value product. Its high value components, such as protein and dietary fiber, have not been utilized as food sources for human consumption, although some health benefits have been reported, such as reduced risk of heart disease ( Daniel D. Gallaher, 17th Annual Distillers Grains Symposium, 2013 year, May 15 to 16 days; http://www.distillersgrains.org/files/scholarships/2013%20Daniel%20Gallaher.pdf).

Similarly, a wide variety of seed meals from oil seeds such as soybean, cotton, sunflower, rapeseed and flax are used in animal feed as a protein source for most livestock. Historically, some oilseeds, such as chia, flax and hemp, have also been consumed by humans because of their claimed health benefits for oils and other ingredients. The oil seed meal of all these crops contains high value proteins mixed with dietary fiber and other ingredients.

However, the feed value of protein is not fully available when oilseed meal is consumed directly for various reasons. The high temperature process used during oil extraction reduces the protein solubility and the nutritional value of the meal. For animals such as fish, chickens and young pigs, high levels of fiber dilute the protein and energy content of the meal and have little feed value. Furthermore, antinutritional factors contained in oilseed biomass, such as trypsin inhibitors and phytic acid, also have an adverse effect. The presence of trypsin inhibitor activity in animal feed reduces growth rate and protein efficiency ratio (PER) (Wilson and Poe, 1985, Aquaculture, 46:19-25). Phytic acid is poorly digested by monogastric species such as pigs, chickens and fish. Phytic acid may complex with minerals, amino acids and proteins, thereby reducing nutrient digestibility. Furthermore, phosphorus in the phytic acid molecule is mainly unavailable to animals and is excreted by feces, causing environmental damage. In general, oilseed meal or waste, when used directly as a feed ingredient, is a protein for monogastric animals such as pigs, chickens and fish due to high fiber, high anti-nutritional factors and high phytate content. The value of feed as a source is limited.

The development of simple green technology to separate expensive proteins from these biomass has great potential in industrial applications.

Various water treatment systems and techniques have often been developed and commercialized to produce high-value protein concentrates and protein isolates from oil seeds such as soybean. However, almost all existing processing systems and technologies focus on producing a single high-value protein product, with little consideration given to the value of the fiber-rich, non-protein components of the starting material, Or not considered. In addition, current technology and processing systems for producing a single high-value protein product from oil seeds often use large amounts of water and chemicals, such as salt, to improve the efficiency of protein extraction and isolation. Consume acid or base. In addition to water and chemical costs, the disposal of low-value by-products or waste streams also has additional costs.

In US Pat. No. 5,658,714, protein is first extracted from vegetable flour by adjusting the pH of the extraction medium to alkaline conditions, and after concentration, the extracted protein is adjusted to pH 3 of the ultrafiltration permeate. A method of precipitating by adjusting to 0.5 to 6.0 is described. US Pat. No. 4,420,425 describes a water extraction process for defatted soybeans using alkaline conditions. After removing the solids by filtration, the solubilized protein extract is concentrated by ultrafiltration with a molecular weight cut off greater than 100 kD to produce a protein concentrate. US Pat. No. 5,989,600 describes a method for improving the solubility of vegetable proteins with enzymes such as phytase and/or proteolytic enzymes. US Pat. No. 3,966,971 teaches a method for extracting vegetable proteins by using acid phytase in an aqueous dispersion.

Various procedures have been reported for the isolation of either dietary fiber or protein constituents from a single product using either chemical or enzymatic methods. Ma et al. (1997, Food Research International, 29(8): 799-805) isolated okara protein by alkaline extraction and isoelectric precipitation. After drying and defatting, the product obtained contains 83% protein. However, isolated proteins have reduced solubility compared to commercial products, which limits their usefulness in the food industry. Solubility can be improved by extended acid denaturation methods (Chan and Man, 1999, Food Research International 32:119-127), but this two-step procedure (protein isolation and subsequent chemical Modification) results in an increase in manufacturing costs and thus reduces the potential for commercialization. Moreover, this method only recovered 53% of the protein in okara and no fiber-rich ingredients were used. A combination of protease and phytase was used to improve the solubility of soy proteins (Bae et al., 2013, J. Food Biochemistry, 37:511-519). However, the method reported begins with the isolation of soy protein and only increases the solubility of the product in the acidic pH range.

Various procedures for making okara fiber have been reported. In one such study (Tian et al., 2007, China Oils and Fats, 32(9):64-66), moist okara was dried, crushed into powders and immersed in an alkaline solution, followed by Enzymatic hydrolysis, bleaching, ethanol precipitation and drying. Surel and Couplet (2005, J Sci Food Agric 85:1343-1349) reported protease hydrolysis of okara protein for purifying okara fiber. This method involved both hydrolysis and delipidation steps of the protein, either by chemical reagents or lipase hydrolysis. In these processes, the protein content is not recovered and the complex steps add to the manufacturing cost.

The procedures reported for extracting either fiber or protein from okara result in waste of other components, often also involving the application of strong chemical reagents and organic solvents. Furthermore, the products obtained are reported to have functional defects, such as reduced fiber water retention capacity (WHC) or reduced protein solubility. To date, no commercial process has been developed for any of these products, and in particular this soy waste has been fully exploited to produce a fiber-rich product and protein in an integrated gentle manner. No procedure has been reported for producing both rich products.

It is necessary to develop a technology that not only solves the problem of disposing of biomass (eg, okara) waste, but can also maximize the use of expensive components in biomass (eg, okara), especially proteins and fibers. ..

This specification refers to several documents, the entire contents of which are incorporated herein by reference.

The following items 1-43 are provided:
1. A method for producing a protein and/or peptide enriched fraction and a dietary fiber enriched fraction from biomass, comprising:
a) incubating the biomass in an aqueous solution under mild alkaline conditions and at a temperature above about 85° C. to obtain an aqueous slurry;
b) treating the aqueous slurry with a proteolytic enzyme under conditions suitable for the activity of the proteolytic enzyme; and c) obtaining a liquid fraction and a solid fraction from the proteolytic enzyme treated slurry of b). The method, wherein the fraction is enriched in proteins and/or peptides and the solid fraction is enriched in dietary fiber.

2. The method of item 1, wherein the biomass is in a wet form.

3. The method according to item 1, wherein the biomass is in a dry form.

4. A method according to item 3, further comprising a step of grinding the dry biomass before step a).

5. 5. The method of item 4, further comprising passing the milled dry biomass through a mesh, optionally a 50-200 μm mesh or a 100 μm mesh.

6. The method according to any one of items 1 to 5, further comprising a step of degreasing the biomass before step a).

7. 7. The method of any one of paragraphs 1-6, wherein the mild alkaline conditions comprise a pH of greater than 7 to about 11 or less, a pH of about 9 to about 11, or a pH of about 10.

8. The method according to any one of items 1 to 7, wherein step a) is carried out at a temperature of about 90°C to about 100°C.

9. 9. The method of item 8, wherein step a) is performed at a temperature of about 90°C to about 95°C.

10. The method of any one of paragraphs 1-9, wherein step a) is performed for about 15 minutes to about 2 hours, about 30 to about 90 minutes, or about 1 hour.

11. 11. The method according to any one of items 1-10, wherein step b) is carried out at a pH of about 7 to about 11.

12. The method according to any one of items 1 to 11, wherein step b) is carried out at a temperature of about 50°C to about 80°C.

13. 13. The method according to item 12, wherein step b) is carried out at a temperature of about 55°C.

14. 14. The method according to any one of paragraphs 1-13, wherein step b) is performed for about 15 minutes to about 2 hours, about 30 to about 90 minutes, or about 1 hour.

15. 15. The method according to any one of items 1-14, wherein the amount of biomass in the aqueous solution is about 0.5% to about 20% (w/v).

16. Item 16. The method according to any one of Items 1 to 15, wherein the proteolytic enzyme comprises subtilisin.

17. 17. The method of item 16, wherein the subtilisin is from Bacillus licheniformis.

18. Item 18. The method according to any one of Items 1 to 17, wherein step c) includes the step of centrifuging the proteolytic enzyme-treated slurry of b) to obtain a liquid fraction and a solid fraction.

19. 19. The method according to any one of items 1-18, further comprising the step of inactivating the proteolytic enzyme after step b).

20. 20. The method of item 19, wherein the deactivating step is heat inactivating.

21. 21. The method of item 20, wherein the heat inactivation is performed at a temperature of about 80°C to about 100°C for about 5 to about 30 minutes.
22. Further comprising the step of (i) b) treating the proteolytic enzyme-treated slurry with a solution containing divalent cations, and/or (ii) treating the liquid fraction of c) with a solution containing divalent cations. 22. The method according to any one of items 1-21, comprising.

23. Solution _comprises CaCl _2, MgCl _2, MnCl ₂ and at least one of FeCl _2, The method of claim 22.

24. 24. The method according to item 22 or 23, comprising the step of treating the liquid fraction of c) with a solution containing a divalent cation to precipitate phytate, and further comprising the step of isolating the liquid fraction from the phytate precipitate. ..

25. 25. The method according to any one of items 22-24, wherein the solution containing divalent cations is used in an amount of about 1.5 to about 20 x equivalents.

26. 26. The method according to any one of items 1-25, further comprising subjecting the liquid fraction to size exclusion chromatography or filtration.

27. 27. The method according to any one of items 1-26, further comprising concentrating the liquid fraction.

28. 28. A method according to any one of items 1-27, wherein the biomass is grain biomass, plant biomass, distillers dried grains (DDG), soybean biomass, rapeseed meal or flaxseed meal.

29. 29. The method according to item 28, wherein the biomass is soybean biomass.

30. Item 30. The method according to Item 29, wherein the soybean biomass is okara.

31. 31. The method according to any one of items 1 to 30, further comprising the step of drying the liquid fraction to obtain a dried product rich in proteins and/or peptides.

32. 32. The method of item 31, wherein the dry product rich in protein and/or peptide has a residual trypsin inhibitor activity that is at least 50% lower than commercial soy protein concentrate (SPC).

33. 33. The method according to item 31 or 32, wherein the dry product rich in protein and/or peptide has a residual phytate content that is at least 60% lower than the commercial soy protein concentrate (SPC).

34. 34. A method according to item 32 or 33, wherein the commercially available SPC is Arcon® F.

35. 35. The method according to any one of items 1-34, further comprising the step of drying the solid fraction to obtain a fiber-rich dry product.

36. 36. The method of item 35, wherein the fiber-rich dry product has a carbohydrate content of greater than or equal to about 70% and a protein content of less than or equal to about 10%.

37. The following features:
a) greater than 80% water solubility over a pH of about 3 to about 11;
b) a protein and/or peptide content of about 40% or higher;
c) at least 75% of the proteins and/or peptides in the extract have a molecular weight of less than 20 kDa;
d) A protein and/or peptide rich dry biomass extract with reduced trypsin inhibitory activity and phytate content compared to commercial soy protein concentrate (SPC).

38. Carbohydrate A dry biomass extract enriched in proteins and/or peptides according to item 37, having a carbohydrate content of about 20% and/or a lipid content of about 10%.

39. Item 37. A dry biomass extract rich in protein and/or peptide according to Item 37 or 38, which is obtained by the method according to any one of Items 31 to 33.

40. A dry biomass extract rich in fiber obtained by the method according to item 35 or 36.

41. Beverages, cosmetics, foodstuffs, comprising the protein and/or peptide-rich dry biomass extract of any one of items 37-39, and/or the fiber-rich dry biomass extract of item 40. Feed products.

42. A method for producing a beverage, cosmetic, food or feed product, the method comprising: (i) performing the method according to any one of items 31 to 33 to obtain a dry product rich in protein and/or peptide. And (ii) a step of incorporating the protein and/or peptide rich dry product into a beverage, cosmetic, food or feed composition.

43. A method of producing a food or feed product, the method comprising: (i) performing a method according to item 35 or 36 to obtain a fiber-rich dry product; and (ii) obtaining the fiber-rich dry product. A method comprising incorporating into a food or feed composition.

Further features will be explained or will become apparent in the course of the detailed description which follows.

FIG. 6 is a schematic diagram showing the method described herein using Okara. Figure 3 is a graph showing Coomassie blue stained SDS-PAGE gel showing the effect of protease E1 enrichment on soybean meal hydrolysis. Figure 3 is a graph showing the effect of Protease E1 enrichment on protein release of soybean meal and okara extract. Figure 3 is a graph showing protein content in supernatants and pellets after various extraction steps. 3 is a graph showing the influence of pH and temperature on protein extraction. 3 is a graph showing the effect of pH and temperature on carbohydrate extraction. FIG. 6 is a graph showing the effect of various proteases on the activity of trypsin inhibitor in Okara extract. NE = no enzyme; enzymes E1-E10 are described in Table 4 below. Figure 3 is a graph showing the effect of various proteases on trypsin inhibitor activity in soybean meal (SBM). NE = no enzyme; enzymes E1-E10 are described in Table 4 below. FIG. 6 is a graph showing the effect of various concentrations of CaCl _{2 on} the phytate content of okara peptide extract. 3 is a graph showing a comparison of solubilities of Okara peptide extract produced by Protease E1 and a commercial product Alcon (registered trademark) F. Fig. 2 is an image showing Coomassie blue stained SDS-PAGE gels of okara and soybean protein extracts. FIG. 6 is an image showing Coomassie blue stained SDS-PAGE gels of hydrolysis supernatants from various biomasses.

DETAILED DESCRIPTION OF THE INVENTION The terms "a" and "an", "the" and similar terms in the context of describing the invention, especially in the context of the claims below. Use of the directives given herein should be construed to cover both the singular and the plural unless specifically stated otherwise herein or clearly inconsistent with the context.

The terms "comprising," "having," "including," and "containing" are to be construed as non-limiting terms unless specifically stated (ie. , "Including but not limited to").

The recitation of ranges of values herein is intended only as a shortened method of referencing each of the individual distinct values of that range unless specifically stated otherwise herein. , Each distinct value is incorporated herein as if individually recited herein. All subsets of values within that range are also incorporated herein as if individually recited herein.

All methods described herein may be performed in any suitable order, unless stated otherwise herein or clearly contradicted by context.

Any and all examples or uses of the terminology (eg, “like”) as used herein are intended only to better describe the invention and are specifically referred to. Unless otherwise stated, the scope of the present invention is not limited.

The term “about” as used herein has its ordinary meaning. The term “about” means that a value includes the intrinsic variation of the error for the device or method used to determine the value, or a value that is close to the listed value, such as the listed value (or the value). Range)) to be included within 10% or 5%.

Any and all combinations and subcombinations of the embodiments and features disclosed herein are encompassed by the present invention. For example, all combinations and subcombinations of the various embodiments of conditions (temperature, pH, time, etc.) with which the method described in the present invention can be carried out are encompassed by the present invention.

The inventors have developed an enzymatic method that directly hydrolyzes proteins from raw biomass after a pretreatment step. Performing a mild alkaline pretreatment at high temperature (eg, about 60 minutes, about 90-95° C., pH about 10), which allows sterilization of the raw material and thus the risk of contaminating later enzymatic processes. Was also minimized and solubilization was promoted, enzymatic accessibility of protein and fiber biomass was increased, resulting in good protein recovery and purified fiber components. Following pretreatment, temperature and pH are adjusted to suit protease function. Following an enzymatic reaction (eg, about 60 minutes), most proteins have been shown to be efficiently hydrolyzed to peptides and/or amino acids of less than 20 KDa, separating the solid fraction from the liquid fraction. did it. Hydrolysis with selected enzymes showed the result of reduced trypsin inhibitory activity in soluble products. Soluble products from Okara contain much less phytate than commercial soy protein concentrate (SPC), further reducing the phytate content in the soluble products and precipitating most residual phytate, if necessary. It was possible to obtain using a divalent cation solution such as CaCl ₂ . The solid fraction was dried to give a fiber rich product and the liquid portion was concentrated and dried to give a protein/peptide rich product.

Accordingly, the present invention produces a protein and/or peptide enriched fraction and a dietary fiber enriched fraction from biomass comprising:
a) incubating the biomass in an aqueous solution under mild alkaline conditions and at a temperature above about 85° C. to obtain an aqueous slurry;
b) treating the aqueous slurry with a proteolytic enzyme under conditions suitable for the activity of the proteolytic enzyme; and c) obtaining a liquid fraction and a solid fraction from the proteolytic enzyme treated slurry of b);
And the liquid fraction is enriched in proteins and/or peptides and the solid fraction is enriched in dietary fiber.

Development of methods for extracting high value products from biomass such as okara and soybean meal
Soybean biomass okara is widely available in waste form from tofu or soy milk factories as waste and also as a dry product, and is now mostly used in the feed industry, with only a few food products. Used in industry. Soybean meal is also widely available from oil seed processing plants after oil extraction. Extraction of such oils may be performed as either thermomechanical or solvent extraction.

Other Biomass The methods developed herein include plant biomass, cereal biomass, other biomass containing significant amounts (about 10% or more) of protein mixed with carbohydrates, and aquatic plants such as duckweed. And seaweed and other types of biomass including microbial biomass such as algae, yeasts, fungi and bacteria may be suitable for extracting proteins/peptides and/or fibers. As shown in the examples below, this method involves soybean biomass (SBM, okara), as well as dried distillers grains (DDG), rapeseed meal, flaxseed meal, whole hemp seeds and peeled hemp seeds. Has been successfully applied to other biomasses including. Optimum reaction conditions (eg pH, temperature and time) need to be adjusted depending on the biomass, but were within the ranges defined herein. For all biomasses tested, the protein component was effectively hydrolyzed in a short time and protein recovery was significantly increased after treatment with this method. For some samples, the protein content in the extracted product was also increased.

In one embodiment, the biomass is in wet form. In another embodiment, the biomass is in dry form.

Conditions for water extraction process (AEP):
Based on conventional water extraction process, using okara and soybean meal as main starting biomass with water suspension in neutral or alkaline conditions, various factors for extraction of soluble fraction-containing protein as main component Was tested for its effect. These factors include: particle size of starting material, extraction time and temperature; extraction liquid conditions: pH adjustment and buffer system.

With regard to the particle size of the starting material, we have found that even coarse grinding to break up large debris may be preferred and no fine grinding step is required. For example, in the case of soybean meal, minimal grinding with a particle size above 1 μm has an effect similar to fine grinding up to 0.125 μm, as well as with dry okara, a simple grinding with a particle size of around 1 μm. Fine milling is equally good as compared to finer milling.

Particularly in the case of soybean meal, it may be preferable to reduce the size of the solid soybean particles by grinding in order to increase the extraction efficiency. The soybean meal has particles of No. It is typically ground to a size that allows it to pass through a 100 mesh (American standard) screen.

Therefore, in one embodiment, the method as defined herein further comprises the step of grinding the biomass (dry biomass) before step a). In a further embodiment, the method comprises milling particles having a size of less than about 200, 150, 100, 50, 25 or 10 μm, such as a milled dry biomass mesh, preferably a 50-200 μm mesh, and more. The method further comprises the step of isolating, preferably by passing through a 100 μm mesh.

In one embodiment, the method as defined herein further comprises the step of defatting (eg removing oil) the biomass prior to step a). Methods of defatting biomass such as soybean biomass (Okara) are known in the art. Degreasing may be accomplished, for example, by chemical extraction using a suitable solvent or by lipase hydrolysis.

Regarding the effect of extraction time on protein yield, it was found that at room temperature, the release of protein gradually increased over time and generally remained constant after about 60 minutes. Thus, in embodiments, the pretreatment step is carried out for a period of less than about 2 hours, or less than about 90 minutes, or less than about 75 minutes. In other embodiments, the pretreatment step is performed for a time of at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes or at least 45 minutes. In a further embodiment, the pretreatment step is carried out for a time of about 15 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 45 minutes to about 75 minutes, about 50 minutes to about 70 minutes, or about 60 minutes. To be done.

Examination of the extraction temperature revealed that up to 90° C. tested, higher temperatures generally resulted in higher extraction rates than room temperature (eg, about 20-25° C.), including soybean meal and okara. Consistent results were obtained for both extractions. Thus, in embodiments, the pretreatment step is performed at a temperature of at least about 85°C, at least about 86°C, at least about 87°C, at least about 88°C, at least about 89°C or at least about 90°C. In embodiments, the pretreatment step is performed at a temperature of about 85°C to about 100°C, about 85°C to about 95°C, or about 88°C to about 92°C, such as about 90°C.

The effect of pH on the extraction of soy protein was tested and the lowest solubility occurs at acidic pH (about 3.5-4), which increases the solubility as it deviates from this pH range and the highest solubility due to mild alkaline conditions. It turns out that Furthermore, the proteins extracted at different pHs showed different composition profiles, as shown by SDS-PAGE. In particular, extraction at pH 2 increased total protein but decreased high molecular weight species. The extraction of both proteins from soybean meal and okara when suspended in either water or buffer (pH 8 0.03M Tris-HCl) was investigated, the buffers yielding protein extraction yields for both biomasses. It was found to increase significantly. However, further analysis comparing a buffer with an aqueous suspension adjusted to a different pH (with NaOH) prior to extraction showed that the increase in protein extraction rate was mainly due to the increase in pH. The liquid-treated biomass showed the same extraction efficiency compared to the water suspension adjusted to the same pH.

Thus, in embodiments, the pretreatment step is carried out at a mildly alkaline pH, eg, a pH of about 7 to about 11, about 8 to about 11, about 9 to about 11, or about 10. The aqueous solution may be, for example, water, a saline solution or a buffer (eg Tris buffer).

The biomass concentration in the aqueous solution for the pretreatment step may be any concentration suitable for carrying out the method, for example at least about 0.1%, at least about 0.5%, at least about 1 %, at least about 2%, at least about 3%, at least about 4%, at least about 5%, or at least about 10% (w/v). In embodiments, the concentration of biomass in the aqueous solution is about 0.1% to about 40%, or the concentration of biomass in the aqueous solution is about 0.5%, about 1%, about 2%, about 3. %, about 4%, about 5%, or about 10% to about 40%, about 30%, or about 20%.

The effects of both combined pH and temperature were tested and it was found that increasing temperature increased protein extractability over the entire range of pH tested. Protein extraction is very efficient when the reaction conditions are set at pH about 8-10 and about 80-90°C. High temperature and high pH conditions can also increase the solubility of carbohydrates, thus reducing the total protein content in the soluble fraction. The effect of combined temperature and pH on total carbohydrate release was analyzed. Between pH 4-11, carbohydrate release was low and was not significantly affected by either pH or temperature. At lower pH, sugar release was significantly increased at all temperatures tested. When the pH was higher than 11, the increased sugar release was measured only at the highest temperature tested. Thus, reaction conditions of pH about 8-10 and about 80-90°C are preferred for good quality and for good yields of protein extraction from okara without increased carbohydrate release. Thus, in an embodiment, the pretreatment is carried out at a temperature of about 88°C to about 92°C, such as about 90°C, and a pH of about 9 to about 11, such as about 10.

To determine how much protein could be released directly from okara without pretreatment (pH 10 and 90° C.), okara was directly extracted 3 times with water and the extracted proteins were combined. Very low amounts were released (7-8%) compared to the total protein in the starting material. In a separate experiment, the okara water suspension was pretreated (preincubation) at 90° C., pH 10 for 1 hour and then washed 3 times. The preincubation step increased recovery of soluble protein. However, only 33% of the total protein in okara was extracted in soluble form. Therefore, next, it will be evaluated whether the enzyme recovery can further improve the protein recovery rate.

Enzyme-assisted water extraction method (EAEP) for extracting products from soybean biomass
The effect of cellulase, hemicellulase, cellobiase, amylase and lipase on the protein extraction efficiency of oilseed biomass was tested using soybean meal (SBM) and okara. Cellulase (Spezyme™ CP), pectinase (Pectinex™ U) and xylanase (HTX4) all increased sugar release. The combination of the three enzymes resulted in even higher sugar release. Treatment with amylase and lipase did not result in sugar release. However, treatment with these enzymes did not significantly increase protein release from biomass.

The effect of protease on total protein recovery was then evaluated. A commercially available protease (alkaline protease, CAS number: 9014-01-1, referred to herein as protease E1) was selected to test this method. When soybean meal and okara were suspended in reaction buffer (pH 8, 0.03M Tris-Cl) and treated with protease E1 (55° C., 1 hour), a very low dose (0.025% v/v) Even the enzyme of (1) could effectively hydrolyze proteins into short peptides directly from raw biomass in a short time. Surprisingly, protease treatment also significantly increases the extracted protein content compared to the no enzyme control incubated under the same conditions. A more detailed analysis was performed using Okara, and the extracted protein increased by 66% as compared to the control without enzyme.

Direct addition of the enzyme to the suspended biomass resulted in an increase in the above protein extraction. It was further investigated whether a combination of pretreatments before enzyme addition could further improve the protein extraction. Pretreatment was carried out by suspending the biomass in the enzyme reaction buffer (pH 8) at the same temperature as the enzyme reaction (55°C). After solid-liquid separation, the released protein in the supernatant is treated as baseline protein release without enzymatic treatment. The pellet was further resuspended and treated with enzyme to see if more protein could be released compared to the sample treated without enzyme. When a pretreatment step was introduced and the pellet was subsequently treated with an enzyme, the D. A simple measurement of 280 released a significant amount of easily detectable protein. Subsequent protease treatment further improved protein recovery. An 89% increase in extracted protein was achieved compared to the sample treated without enzyme. However, the total protein recovery was only about 30% and the majority of the protein remained in the pellet insoluble fraction. Pretreatment was performed at higher temperature (90° C.) and pH 8 for 1 hour, followed by enzyme treatment, which resulted in 59% total protein recovery compared to 38% recovery without enzyme treatment. Achieved Pretreatment was performed at 90° C. and pH 10, followed by enzyme treatment, achieving total protein recovery from okara biomass of up to 85%. The protein content in the fiber-rich insoluble fraction was significantly reduced. These results show that a combination of mild alkaline conditions (pH 10) and pretreatment at elevated temperature, followed by a treatment step with protease enzymes is efficient for protein extraction. Moreover, such enzyme-assisted water extraction methods not only increase the total extraction efficiency of the protein, but may also have a better function, eg, a peptide product that may have an increased nutrient digestibility compared to the full length protein/peptide mixture. Can have a twofold benefit.

The activity of 10 different proteases (see Enzymes E1 to E10 in Table 4 below) on standard substrates was characterized and for soy protein hydrolysis profiles using standardized enzyme doses, soybean meal and okara. Both were analyzed. The present inventors have found that Bacillus licheniformis (serine type protease, subtilisin, Sigma (registered trademark) Cat. No. P5459 and EMD Millipore Cat. No. 126741), Bacillus amyloliquefaciens. Serine type protease, subtilisin, Sigma (registered trademark) Cat. No. P1236), Aspergillus oryzae (endoprotease and exopeptidase, Sigma (registered trademark) Cat. No. P6110), genus Bacillus (Bacillus). (A serine-type protease, subtilisin, Sigma (registered trademark) Cat. Nos. P3111, P5985, P5860) and various proteases including cysteine proteases (papain, Sigma (registered trademark) Cat. Nos. P3375 and P76220). It was used and found to produce a similar hydrolysis pattern. In most hydrolysis processes, proteins and peptides of all sizes were gradually reduced by a rather early accumulation of short peptides. For all enzymes tested, the majority of hydrolyzed peptides had a molecular mass of less than 30 kDa.

Based on SDS-PAGE analysis, most of these enzymes appear to release peptides in a range of similar molecular sizes, but the yields and identities of these peptides are not the same. There is. For example, the physical and nutraceutical functions of peptides from different protease hydrolysis may not be the same due to different composition. One of ordinary skill in the art can select the protease (or combination thereof) to be used in this method based on the desired criteria, eg, better hydrolysis efficiency under certain conditions, desired activity, etc. You will understand.

Ten proteases were tested for their ability to produce products with reduced trypsin inhibitory activity. Compared to the material treated without enzyme, treatment with different enzymes reduced trypsin inhibition to varying degrees, the difference resulting from the different composition of the hydrolyzed products obtained. Seem.

Thus, in embodiments, the methods described herein include endoproteases, exopeptidases, serine-type proteases (eg, subtilisin), cysteine-type proteases (eg, papain), threonine-type proteases, asparagine-type proteases, glutamine-type proteases, metallo-proteases. It can be performed using any proteolytic enzyme (protease), including proteases and asparagine peptide lyases, or a combination thereof. These proteases can be isolated or recombinantly produced from any suitable organism (bacteria, fungi, plants, animals, etc.) using commonly used techniques. In one embodiment, the method as defined herein comprises the use of one or more serine-type proteases, such as subtilisin. In another embodiment, the method as defined herein comprises the use of one or more cysteine type proteases such as papain. In another embodiment, the method as defined herein comprises the use of one or more of the enzymes E1-E10 listed in Table 4. In one embodiment, the starting biomass is okara and the method as defined herein comprises the use of one or more of the enzymes E1, E3, E8 and E10 listed in Table 4. In one embodiment, the starting biomass is SBM and the method as defined herein comprises the use of one or more of the enzymes E3, E4 and E9 listed in Table 4.

Conditions for the proteolytic step (eg temperature, pH, time) may be adapted based on the protease(s) used. In one embodiment, the temperature of the proteolysis step is about 20°C to about 80°C, about 30°C to about 70°C, about 40°C to about 60°C, or about 50°C to about 60°C. In embodiments, the proteolytic step is performed at a pH of about 4 to about 12, about 7 to about 11, about 8 to about 11, about 9 to about 11 or about 10. The proteolysis step may be performed in the same aqueous solution as the pretreatment step or in a different solution. The pH and temperature may be readjusted between the pretreatment step and the proteolysis step.

In embodiments, the proteolysis step is performed for a time of less than about 2 hours, or less than about 90 minutes, or less than about 75 minutes. In other embodiments, the proteolytic step is performed for a time of at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes or at least 45 minutes. In a further embodiment, the proteolytic step is performed for a time of about 15 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 45 minutes to about 75 minutes, about 50 minutes to about 70 minutes, or about 60 minutes. To be done.

In one embodiment, the method as defined herein further comprises the step of inactivating the proteolytic enzyme after the proteolytic step. Methods of inactivating proteases are well known in the art, eg, chemical inactivation (eg, using protease inhibitors), pH inactivation (eg, pH of mixture/slurry is incompatible with proteolytic activity). By adding an acid or a base) so that heat deactivation is achieved. In one embodiment, the step of inactivating the proteolytic enzyme comprises heat inactivation, for example by heating the slurry to a temperature of at least 70°C or 80°C for at least 5 or 10 minutes. In one embodiment, heat quenching involves heating the slurry to a temperature of 80° C. to about 100° C. (eg, 80° C., 85° C., 90° C. or 95° C.) for about 5 minutes to about 30 minutes, preferably about 10 minutes. Including heating for a time of 20 minutes or about 15 minutes.

Reducing the Phytate (Phytic Acid) Content in the Product It may be desirable to reduce the phytate content in the product obtained using the methods described herein. It is clear that phytate is often referred to as a nutritional inhibitor because of its interference with the absorption of certain nutrients, such as minerals (calcium, magnesium, iron, copper and zinc). In order to accomplish this task, were tested (precipitation by) divalent cations selected to reduce the concentration of non-nutritive phytate in peptide product _{(CaCl 2, MgCl 2, MnCl} 2 and FeCl _2). When tested under manufacturing conditions (pH 10), MnCl ₂ appeared to be the most effective at inducing phytate precipitation, especially at lower concentrations. At 1.5x equivalents (1 equivalent, 1x is defined as 6 divalent cations per molecule of phytate eg molecules of calcium) and higher concentrations CaCl ₂ and FeCl ₂ are also very high. Well implemented. CaCl ₂ was able to reduce 95% phytate concentration in the final product when tested in bench scale experiments when used in optimized conditions and higher concentrations.

Thus, in one embodiment, the method as defined herein further comprises reducing the phytate content. Methods of reducing the phytate content in a composition are well known in the art and include, for example, treatment with phytate-degrading enzymes (eg phytase) or divalent cations. The phytase may be from any source/source, eg fungal, bacterial, yeast or plant. In a further embodiment, the method comprises adding the proteolytic enzyme treated slurry of step b) to a solution containing one or more divalent cations, eg one of CaCl ₂ , MgCl ₂ , MnCl ₂ and FeCl _2. Or a step of treating with a solution containing a plurality of species. In a further embodiment, the solution comprises CaCl ₂ . In embodiments, the amount of divalent cations in the slurry is about 0.5x to about 50x equivalents, or about 1x to about 40x equivalents, or about 1.5x to about 25x equivalents.

Final method developed for product recovery and quality Two-step protease method, addition of suitable enzyme candidates for proteolysis and reduced trypsin inhibition activity, and addition of divalent cations for phytate precipitation The conditions were determined and the method was tailored for optimal product recovery and quality. According to the embodiments described herein, the optimal parameters identified are as follows: pretreatment at about 90° C., pH 10 for about 1 hour, followed by treatment with the selected dose of enzyme for about 1 hour. , And CaCl ₂ were added at the specified doses. The supernatant (liquid fraction) is then separated from the solid fraction (eg, using centrifugation, filtration, or any other method suitable for separating the liquid and solid fractions) and concentrated. And dried to yield a protein/peptide rich soluble product. The fiber-enriched solid fraction may be dried for use as a fiber product for food or feed.

Therefore, the present invention provides a method for producing a protein and/or peptide-enriched fraction and a dietary fiber-enriched fraction from biomass,
a) obtaining the biomass in either dry or wet form;
b) optionally grinding the biomass;
c) solubilizing and extracting the biomass or crushed biomass with an aqueous solution having a pH adjusted to alkaline conditions and a temperature adjusted to 90° C. or higher;
d) readjusting the temperature and pH of the aqueous slurry and treating the aqueous slurry with a proteolytic enzyme;
e) optionally reducing the phytate content, for example by adding a divalent cation solution (eg CaCl ₂ ) to the slurry to precipitate the phytate content;
f) optionally inactivating the proteolytic enzyme;
g) separating the liquid and solid fractions;
h) optionally concentrating the liquid fraction to obtain a liquid concentrate;
i) optionally drying the liquid concentrate to obtain a soluble protein and peptide rich product; and j) optionally drying the solid fraction to obtain a fiber rich water insoluble product. A method, including the steps of obtaining.

In embodiments, step e) may be performed after step g), followed by a separate solid/liquid separation to remove the precipitated phytate content. The liquid fraction may be further concentrated and dried as the final product.

In embodiments, prior to step h), the liquid fraction is subjected to one or more purification step(s), for example molecular mass or using a membrane filtration system based on target product characteristics. It may be subjected to purification based on the size difference.

In one embodiment, the method as defined herein does not include the use of organic solvents.

Product Characterization Table 1 shows a compositional comparison of Alcon® F (Archer Daniels Midland, ADM) with the two peptide products obtained by the methods described herein. Amino acid profile analysis of protein/peptide samples extracted from okara showed a ratio of both essential and non-essential amino acids when compared to the commercial soy protein concentrate (SPC) Alcon® F from ADM. Indicate that they are similar, and that if other characteristics are the same, they can expect the same nutritional value. However, the solubility analysis shows that the commercially available SPC Alcon® F exhibits only about 10% solubility between pH 3-9 and rises to 25% only at pH 11, while the method described herein. It was shown that the extracted protein/peptide product obtained by pt. s. showed consistently high solubility (>80%) over a wide range of pH (3-11). Compared to Alcon® F, the peptide product obtained from the okara derived by the method described herein has a lower protein content mainly in the starting material (okara, 25% relative to soybean meal, usually (>50%) and lower amounts of protein due to excess lipid in okara and peptide products. However, lower concentrations of anti-nutritional factors were detected in the okara extract obtained by the method described herein, as compared to Alcon® F. Enzymatic methods also resulted in protein/peptide products with a degree of hydrolysis above 20% and mostly below 20 kDa. This method also resulted in lower trypsin inhibitory activity and phytic acid content. Thus, the extracted protein/peptide product obtained by the method described herein can be used in a wide range of applications including foods, feeds, beverages, cosmetics, and has good function and nutritional value.

Accordingly, in another aspect, the invention relates to a protein and/or peptide rich dry biomass extract, preferably soybean biomass extract, comprising one or more of the features described herein. In one embodiment, the protein and/or peptide rich dry biomass extract comprises at least 2, 3, 4, or 5 of the features described herein. In a further embodiment, the extract has the following characteristics:
a) greater than 80% water solubility over a pH of about 3 to about 11;
b) a protein and/or peptide content of about 40% or higher;
c) at least 75% of the proteins and/or peptides in the extract have a molecular weight of less than 20 kDa;
d) Contain at least one, two, three or all of reduced trypsin inhibitory activity and phytate content compared to commercial soy protein concentrate (SPC).

In a further embodiment, at least 80%, at least 85%, or at least 90% of the proteins and/or peptides in the extract have a molecular weight of about 20 kDa.

In one embodiment, the extract has a trypsin inhibitory activity of less than about 3, 2.5 or 2 TUI/mg as measured using the method described in the Examples below.

In one embodiment, the extract has a phytate content of less than about 25 or 20 mg/g, measured using the method described in the Examples below.

In one embodiment, a dry biomass extract rich in proteins and/or peptides is obtained by the methods described herein.

In another aspect, the invention provides a fiber-rich dry biomass extract, preferably a soybean biomass extract, comprising one or more of the features described herein. In one embodiment, a fiber-rich dry biomass extract is obtained by the methods described herein.

In embodiments, the extracts described herein may be used in various food products, such as beverages (eg, soft drinks), dairy products, sauces, baked goods, nutrition bars, cereals, confectionery such as candies, gums, jellies, and the like. , Tablets, bread, cooked rice, vegetarian foods (hamburgers, sausages, granola products, putties), etc. In one embodiment, the extracts described herein are incorporated into animal feed (livestock, pets).

In embodiments, the extracts described herein can be incorporated into cosmetic products/compositions. Such cosmetic products/compositions can be, for example, in the form of creams, emulsions, foams, gels, lotions, milks, mousses, ointments, pastes, powders, sprays or suspensions. The cosmetic product/composition optionally comprises at least one cosmetically acceptable auxiliary agent. Cosmetically acceptable auxiliaries include carriers, excipients, emulsifiers, surfactants, preservatives, fragrances, perfume oils, thickeners, polymers, gel formers, dyes, absorption pigments, light protectants, viscosity modifiers. Agent, antioxidant, defoaming agent, antistatic agent, resin, solvent, dissolution promoter, neutralizing agent, stabilizer, bactericide, spray, desiccant, opacifying agent, cosmetic active ingredient, hair polymer, hair Conditioner agents and skin conditioner agents, graft polymer agents, water-soluble or dispersible silicone-containing polymer agents, bleaching agents, care agents, coloring agents, colorants, tanning agents, water retention agents, re-fatting agents, collagen, protein hydrolysis Substances, lipids, emollients and emollients, colorants, sunscreens, bleaches, keratin hardening substances, antibacterial active ingredients, optical filter active ingredients, repellent active ingredients, hyperemic substances, keratolytic agents and keratin formation Keratoplastic substance, antidandruff active ingredient, anti-inflammatory agent, keratinizing substance, active ingredient acting as antioxidant and/or active ingredient acting as free radical scavenger, skin moisturizing substance or skin water retentive substance, refat Examples thereof include, but are not limited to, emollient active ingredients, deodorant active ingredients, sebostatic active ingredients, plant extracts, antierythematous or antiallergic active ingredients, and mixtures thereof.

The invention also relates to a beverage, cosmetic, food or feed product comprising a dry biomass extract enriched in proteins and/or peptides as described herein or a dry biomass extract enriched in fibers.

The present invention also includes (i) performing the method described herein to obtain a dry product enriched in proteins and/or peptides, and the dry product enriched in proteins and/or peptides in beverages, cosmetics. , A method of manufacturing a beverage, cosmetic, food or feed product, comprising the step of incorporating into a food or feed composition.

The present invention also includes (i) performing the method described herein to obtain a fiber-rich dry product, and incorporating said fiber-rich dry product into a beverage, cosmetic, food or feed composition. A method of manufacturing a beverage, cosmetic, food or feed product comprising steps.

In order to make the present invention more clearly understood, its embodiments will be described in more detail by using non-limiting examples with reference to the accompanying drawings.

Example 1: Dose Analysis of Protease E1 for Okara and Soybean Meal Hydrolysis To analyze whether proteases can efficiently hydrolyze soy proteins directly from raw biomass without first protein purification. A freeze-dried defatted soybean meal sample was suspended in 0.03 M Tris-HCl (pH 8.0) buffer at a 2.8% solids:liquid ratio (W/V) and enzyme was added. Incubated at 55°C for 1 hour. One of the common commercially available proteases (Sigma P5459; defined herein as Protease E1) was used to test the quantitative effect on soybean protein hydrolysis. For the blank, the reaction was carried out in a separate tube without addition of enzyme. The enzyme doses tested were 0.0025%, 0.005%, 0.01%, 0.02%, 0.04%, 0.08% (v/v). After 1 hour, the reaction was stopped by incubating the reaction tube at 95°C for 15 minutes. The reaction tube was centrifuged at 20,000 xg for 20 minutes to separate the liquid and solid fractions. The supernatant was analyzed by SDS-PAGE gel. An equal volume (15 μl) of liquid from each reaction was loaded onto a 12% acrylamide gel and an SDS-PAGE gel was run at 130 volts for 1 hour, then stained with Coomassie blue. These results are presented in Figure 2. Very low doses of enzyme were sufficient to hydrolyze the higher molecular weight proteins to predominantly less than 40 kDa compared to the no enzyme control. Increasing the dose further reduced the density of higher molecular weight proteins, as well as the density of lower molecular weight proteins of approximately 30-40 kDa, which meant that more protein was hydrolyzed, It has been suggested that it resulted in smaller peptides of less than 30 kDa. At a dose of 0.04%, the majority of proteins were hydrolyzed into short peptides, except for the fraction of very weak peptides of approximately 30-40 kDa. When the dose was further increased to 0.08%, the approximately 30 kDa peptide became almost invisible. These results suggest that soy protein was extensively hydrolyzed by small doses of protease E1 into short peptides or amino acids within 1 hour.

To analyze the quantitative effect of Protease E1 on protein extraction from soybean and okara, both soybean meal and okara samples were freeze-dried and placed in 0.03M Tris-HCl (pH 8.0) buffer. Suspended at a solid: liquid ratio (W/V) of 0.8%. Incubation of the reaction mixture, protease E1 dose, and solid-liquid separation were the same as above. O. D. Supernatants were tested for released protein content by measuring the absorbance at 280 nm. O. D. The 280 values were plotted against enzyme dose (Figure 3). For both okara and soybean meal, the enzyme affects protein release at very low doses. As the dose increased, the release of protein increased, which became constant at a dose of about 0.05%. This significant increase in the release of either soybean meal or okara protein due to protease hydrolysis was unexpected. Further detailed characteristics of this effect are reported in the examples below.

Example 2: Effect of Protease on Protein Extraction Yield from Okara To further determine the effect of protease treatment on protein extraction from okara, freeze-dried okara was treated with 0.03 M Tris-HCl (pH 8). 0.0) Suspended in buffer at 2.8% solids: liquid ratio and incubated for 1 hour at 55°C. Solids and liquids were separated on a bench top centrifuge by centrifugation at 20,000 g for 10 minutes and the supernatant retained. Protease E1 was added at a ratio of 0.01% v/v and the reaction mixture was incubated at 55° C. for 1 hour. The reaction was then stopped by incubating at 95°C for 15 minutes. For reactions without enzyme, separate reactions without enzyme addition were run in parallel. After a 15 minute step at 95°C, the supernatant was collected after centrifugation and the pellet was extracted again at 55°C with the same buffer for 1 hour. After liquid and solid separation, the protein content of all separately retained and freeze-dried liquids was determined by using the Bradford Protein Assay (BPA) method with the Coomassie Plus Assay Kit (Thermo Scientific) according to the manufacturer's procedure. analyzed. Alternatively, the okara sample was suspended in Tris buffer and treated directly with protease, followed by a wash step as described above. A parallel enzyme-free control experiment was performed in separate reactions. The proteins released in the supernatant from each step were analyzed using the same kit. These results are summarized in Table 2.

When the protease was added directly to the okara suspension, the amount of protein extracted in the enzyme step was twice that extracted in the no enzyme control. When the post-treatment step was included, the total protein extracted was increased by 66% compared to the no enzyme control. When the pretreatment step was introduced, the enzyme treatment step alone increased the protein extraction by 4.5-fold compared to the no enzyme control. An 89% increase in total protein extracted was achieved when total protein from all three steps was combined.

Example 3: Effect of Protease on Soluble Form and Oat Protein Content of Fiber Pellets All the protein content for solid biomass, okara, fiber pellets and extracted soluble fractions was determined by Gerhardt Kjeldatherm Degradator. , Gerhardt Vapodest(TM) 20s distillation apparatus using the Kjeldahl method (AOAC Official Methods 2001.11; J AOAC Int. 1999, 82:1389-1398), and SI analysis titroline (Titroline). ) (Trademark) 6000.

To determine the protein that released water, freeze-dried okara (250 mg) was suspended in water at room temperature at a 2.8% solids:liquid ratio. Solids and liquids were separated by an Allegra(TM) X-12R (Beckman Coulter) centrifuge by centrifugation at 2800 g for 15 minutes and the supernatant was saved. The pellet was resuspended and this method was repeated two more times. Supernatants from 3 replicates were combined. Both pooled supernatant and pellets were freeze-dried and protein content was determined by the Kjeldahl method.

For okara pre-incubation and washing, freeze-dried okara was suspended as in the previous step and incubated for 1 hour at pH 8.0 (adjusted with 4N NaOH) and 90°C. The incubation supernatant and the three washes were combined. The protein content of the supernatant and pellet was determined as described in the previous step.

For enzyme blanks, okara was washed 3 times and incubated at 90° C. at pH 8.0 for 1 hour. The liquid was separated from the solid and the pellet was washed 3 times as above. The pellet was then resuspended in water to its original volume, pH adjusted to 8.0 and incubated at 55°C for 1 hour without enzyme. The liquid was separated from the solid as above and the pellet was washed 3 times. All liquid supernatants were combined. Both pooled liquid and washed pellets were freeze dried and protein content was determined by the Kjeldahl method. For enzyme-assisted extraction, the procedure was all the same as the enzyme blank except the addition of 0.005% (V/V) protease E1 at 55° C. for 1 hour.

The protein recovery in the supernatant and the residual protein content in the pellet after various manipulations are shown in Table 3 and FIG. Three washes at room temperature resulted in 10% protein release; hot pH 8 pre-incubation followed by 3 washes resulted in 33% protein recovery; another step (at 55°C) 38% protein recovery was achieved when enzyme-free treatment was added for 1 h) followed by 3 washes; 59% by adding enzyme at 55° C. for 1 h step. Protein recovery was obtained in the supernatant. Correspondingly, the protein content remaining in the pellet was reduced from 87% to 36%.

Example 4: Effect of pH and temperature on protein extraction Freeze-dried okara was suspended in water at a 2.5% (W/V) ratio and the pH was adjusted to 4N HCl (pH 1.5→7) or 4N. Of NaOH (pH 7→12). Okara suspensions of different pH were incubated at different temperatures for 1 hour. Solid and liquid separation was done by centrifugation at 2800 g for 10 minutes. The protein content in the supernatant was analyzed using the Protein DC kit (BioRad®) according to the manufacturer's procedure.

The effect of combined pH and temperature is shown in the graph depicted in FIG. Increased temperature increased protein extractability over the entire pH range tested. Protein solubility was lowest between pH 3-5 and increased in both directions when pH was adjusted to either higher or lower. At 90°C, pH 1 and pH 10 resulted in similar high protein release. High protein recovery at pH 10 and 90°C, these conditions would be compatible with the actual manufacturing method.

Example 5: Effect of pH and temperature on carbohydrate extraction Total carbohydrates were analyzed by the modified phenol/sulfuric acid method (Nielsen, 2003, Food Analysis Laboratory Manual, Chapter 6; DuBois et al., 1956, Anal. Chem., 28:350-356; Mecozzi, 2005, Chemometrics and Intelligent Laboratory Systems, 79:84-90). Okara samples were suspended, incubated at different temperatures and pH, and extracted as above for 1 hour. Supernatants (10 μL) from different extractions were placed in 15 mL Falcon™ tubes. The raw material H ₂ SO ₄ was added directly to the tube after addition of water and 80% phenol. The sample was vortexed and allowed to stand at room temperature for 10 minutes, then cooled in a 25° C. water bath for 10 minutes, followed by O.V. D. 490 readings were taken. O. D. Values versus pH were plotted as shown in Figure 6.

One major goal for method development has been to increase protein recovery and protein content in extracted products. Protein content is also affected by the released carbohydrates in the extracted product. The effect of both combined pH and temperature on sugar release was tested. Between pH 4-11, carbohydrate release was low and was not significantly affected by either pH or temperature. At lower pH, sugar release was significantly increased under all temperatures tested. At pH higher than 11, the sugar release was only measured to increase at the highest temperature tested. Thus, even with high protein release, acidic conditions would not be preferred. At higher pH and temperature, carbohydrate release does not increase, while protein release increases, thus allowing for increased protein recovery and higher protein content in the extracted product.

Example 6: Effect of enzyme treatment on trypsin inhibitor activity of soybean samples The ten commercially available proteases (E1-E10) listed in Table 4 were established using okara and defatted soybean meal. Tested according to the EAEP method. The relative activity of proteases was tested according to the general protease activity assay procedure (Sigma®) with the non-specific substrate casein (Sigma®). Pre-treated okara and soybean meal (SBM) were hydrolyzed with standardized amounts of each enzyme.

The trypsin inhibitor activity assay was performed according to published methods with minor modifications (Kakade et al., 1969 Cereal Chem 46:518-526; Kakade et al., 1974, Cereal Chem 51:376-381). The dried sample (0.5 g) was ground, passed through a 60 mesh sieve and extracted with 25 ml of 0.01 N NaOH for 3 hours with shaking at 150 rpm at room temperature (suspension pH ca. 9.5-9.8). The suspension was diluted so that 40-60% trypsin inhibition was achieved. After mixing with the trypsin solution, the reaction mixture was incubated in a 37° C. water bath for 10 minutes, followed by addition of the benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPA) solution, for a further 10 minutes. Incubated. After stopping the reaction with acetic acid, O. D. 410 was measured against a reagent blank and a sample blank.

Trypsin units (TU) are optionally defined as the increase in absorbance units at 410 nm of 0.01 per 10 ml reaction mixture under the conditions used. Trypsin Unit Inhibition (TUI) is the difference in trypsin units assayed with and without soybean samples.

All enzyme treatments resulted in varying degrees of reduction of trypsin inhibitory activity, as shown in FIGS. 7A and 7B, as compared to control samples. Enzymes 1, 3, 8 and 10 demonstrated the most significant effect on okara and enzymes 3, 4 and 9 were most effective on soybean meal.

Example 7: Phytate content of proteins extracted from okara The phytate content assay was performed according to published methods with minor modifications (Dragicevic et al., 2011, Acta periodica technology 42: 11-21; Gao et al. , 2007, Crop Science 47: 1797-1803).

The dried okara sample (0.5 g) was passed through a 60 mesh screen and incubated with HCl and TCA for 2 hours at room temperature with shaking at 250 rpm. After centrifugation at 10,000 g at 10° C. for 20 minutes, the supernatant was filtered through a 0.22 μM syringe filter unit, then diluted 25 times with deionized H ₂ O.

Colorimetric determination of phytate content was performed by adding Wade reagent (Wade reagent) (0.03% of FeCl ₃ 6H ₂ O and 0.3% sulfosalicylic acid) to the reaction tubes. After centrifugation, the absorbance was measured at 500 nm.

Calculation: O. D. The reduced value at 500 reflects the phytate content in the sample obtained by subtracting the absorbance of the sample from the reagent blank absorbance. The amount of phytate was calculated by a sodium phytate standard curve.

The effect of CaCl ₂ addition on the phytate concentration in the extracted peptide product from okara was investigated. In separate experiments, different amounts of CaCl ₂ were added during the extraction process and samples were taken at different time points. As shown in FIG. 8, as the equivalent of CaCl ₂ in the hydrolysis process increased, the amount of phytate in the extracted soluble product decreased in a dose-dependent manner. Above 15 equivalents, the phytate content dropped to a minimum 30 minutes after the addition of CaCl ₂ . At the end of this process, the okara control slurry (without CaCl ₂ added) had a phytate of about 240 μg/mL, while adding CaCl ₂ at a higher concentration, FIG. As shown in, the phytate was reduced by up to 95% in the final product.

Example 8: Degree of hydrolysis of okara peptide (% DH)
Okara biomass was suspended in water at a 10% solid:liquid ratio, the pH was adjusted to 10 with 4N NaOH and extracted at 90° C. for 1 hour. Then the temperature was adjusted to 55° C. and the pH was readjusted to 8 using 4N HCl. Protease 1 was added at a dose of 0.005% and hydrolysis samples were taken at different times. The experiment was repeated and a control experiment without enzyme was run in parallel as a comparison. The hydrolysis process was monitored by measuring the degree of hydrolysis (% DH).

% DH was performed with o-phthaldialdehyde (OPA) according to the published procedure with minor modifications (Nielsen et al., 2001, J Food Sci. 66:642-646; Vigo et al. 1992, Food Chem. 44:363-365). Samples were diluted to contain 1-10 mg protein per mL and the dilution factor recorded. After addition of all the reagents, before measuring the A _340, the mixture was just allowed to stand for 2 minutes.
DH was calculated as follows:% DH=h/h _tot ^* 100;
h = (serine _-NH 2 _-β) / _α;
Parameters for soy protein: β=0.342; α=0.970; h _tot =7.8.
Serine -NH ₂ equivalent from OD readings were obtained as follows:

Reaction volume (L), sample weight (g) and protein concentration were obtained based on reaction conditions. Enter the% protein of the sample as a percentage, not as a fraction.

The results, shown in Table 5, show that within 5 minutes of reaction the decrease in hydrolysis increases and the DH continues to increase towards the end. At 60 minutes, a% DH of 24-27% was reached and the control sample remained constant between 7-10%.

Example 9: Solubility Comparison of Okara Products Using the Commercially Available Alcon(R) F Protein solubility was determined by using the method of Lee and Morr with slight modifications ( Lee et al., 2003, JAOCS 80, pages 85-90; Morr et al., 1985, J. Food Sci. 50: 1715-1718). One batch of okara peptide product made with Protease E1 and the commercial product Alcon® F(ADM) was suspended in a 0.1 M NaCl solution at a concentration of 2% (W/V). .. After adjusting the pH with 1N NaOH or 1N HCl solution, the suspension was thoroughly mixed on an air shaker at room temperature and 100 rpm for 30 minutes. The suspension was then centrifuged at 20,000g for 15 minutes. The protein content of the supernatant and the original solid powder was measured using the Kjeldahl method and a conversion factor of 6.25. Protein solubility was calculated as the percentage of protein in the supernatant relative to total protein in the original sample.

The extracted protein obtained by the method described herein, as compared to the commercial soy protein concentrate Alcon® F, which exhibited a solubility of about 10% at pH 3-9 and 25% at pH 11 The /peptide product showed a consistently high solubility (>80%) over the pH range tested (3-11) (Figure 9).

Example 10: With the effect preprocessing step of the enzyme and CaCl ₂ treatment to the concentration of protein and antinutritional factors okara soluble extract, the established procedure and a subsequent enzymatic treatment, CaCl ₂ During the enzyme step Was added in 5 equivalents to reduce the concentration of phytate, which is an antinutrient. Extraction was performed 3 times on a 1 L scale. Control extractions without enzyme and/or without CaCl ₂ were also performed 3 times. A sample of the final extract was lyophilized and analyzed for yield and antinutritional factors (trypsin inhibitory activity and phytic acid). The extraction procedure with enzyme and CaCl ₂ gave a protein recovery of 53%, which is slightly lower than the extraction without CaCl ₂ (60%), which indicates that CaCl ₂ was not It has been suggested that it may reduce solubility or precipitate some soluble proteins when added to a biomass suspension. However, the enzymatic process with or without CaCl ₂ produced a greater protein yield than the extraction without enzyme (47%). Furthermore, the protein content in the hydrolyzate was also increased. In terms of reproducibility, all three extraction procedures yielded varying protein yields of 8.5% (standard deviation) or less. Anti-nutritional factors were reduced in the extracted product when CaCl ₂ and protease were used in this procedure. As shown in Table 6, the addition of CaCl ₂ reduced phytic acid by 40% and the addition of enzyme reduced trypsin inhibitory activity by 60%. Depending on the needs of the final product, the phytate content may be further reduced by increasing the CaCl ₂ concentration if necessary.

Example 11: Comparison of Proteins Extracted from Okara and Whole Soybeans Okara protein was prepared by drying dried okara in Tris-Cl buffer at pH 8 or at various pHs (pH 6, 7, 8 as described above). , 9 and 10) and suspended in water for extraction. Soy milk from the same type of soybeans as okara is first prepared by soaking the dried soybeans in water for 2 hours at room temperature, followed by grinding for 10 minutes and then boiling the suspension of the ground material for 30 minutes. Was prepared. Soy milk was obtained by filtering through three layers of cheesecloth. The extracted samples were compared by performing an SDS-PAGE gel followed by staining with Coomassie blue. The peptide profile of the protein extracted from okara showed a very similar pattern with both stored soybean 11S/7S protein and other minor components (FIG. 10). This suggests that there is no significant difference in protein/peptide composition between Okara extract protein and whole soybean extract protein.

In addition, the amino acid profiles of four different batches of okara peptide extract were compared to the amino acid profile of the commercial soy protein concentrate Alcon® F. Both Arcon® F and okara peptide extract were hydrolyzed with HCl according to conventional methods. Amino acid profile analysis was performed according to the manufacturer's instructions using the Agilent ZORBAX™ Eclipse Plus C18AA method using a Zorbax™ Extend C18 column (Agilent p/n 763954-302) (Agilent). Application Note 5990-454, for foods and pharmaceuticals). The amino acid internal standard preparation, mobile phase and gradient were selected at a flow rate of 0.42 mL/min according to the manufacturer's instructions. Detection with a diode array detector (DAD) and quantification (internal standard and calibration solution) were also performed according to the manufacturer's instructions. The assay was repeated twice for each sample and the average of the replicates is listed in Table 7.

Example 12: Application of the developed method on other biomass The method established on the basis of Okara extraction was tested on other biomass at 100 mL scale; for each biomass, protein recovery and content in the extracted product. It was determined. Protein recovery was determined by comparing the amount of protein extracted to total protein in raw biomass. Protein content was determined by comparing the amount of extracted protein to the total weight of extracted product on a dry matter basis. The pretreatment pH was raised to pH 11 for the extraction of distillers dried grains (DDG) and oilseed rape to increase protein yield. The above procedure was followed for other steps and other samples. As shown in Table 8, for all materials tested, the enzymatic process significantly increased protein recovery. The greatest increase in protein recovery was seen with DDG (40%-74%), followed by rapenamile (65%-87%), as compared to the no enzyme control. In terms of protein content in the extracted product, DDG and rapenamile showed the greatest increase, while for flax and soybean, a slight increase was obtained.

To determine if the previously described method, which is mostly based on okara, can be used to hydrolyze other biomass, a small-scale extraction was carried out with protease E1 to produce okara, soybean meal, DDG and rapeseed meal. Was carried out. For each biomass, the supernatant with and without enzyme was analyzed side by side on a 15% acrylamide gel. As can be seen in FIG. 11, when the various materials were incubated with the enzyme, a conversion of higher to lower molecular weight material was apparent. For okara, most proteins are hydrolyzed to peptides less than 15 kDa, for soybean meal most less than 25 kDa, for DDG most less than 10 kDa, and for canola meal most It was less than 10 kDa.

The scope of the claims should not be limited to the preferred embodiments described in the examples, but should be given the broadest interpretation consistent with the specification as a whole.

Claims (43)

A method for producing a protein and/or peptide enriched fraction and a dietary fiber enriched fraction from biomass, comprising:
a) incubating the biomass in an aqueous solution under mild alkaline conditions and at a temperature above about 85° C. to obtain an aqueous slurry;
b) treating the aqueous slurry with a proteolytic enzyme under conditions suitable for the activity of the proteolytic enzyme; and c) obtaining a liquid fraction and a solid fraction from the proteolytic enzyme treated slurry of b);
Wherein the liquid fraction is enriched in proteins and/or peptides and the solid fraction is enriched in dietary fiber. The method of claim 1, wherein the biomass is in wet form. The method of claim 1, wherein the biomass is in dry form. The method of claim 3, further comprising the step of comminuting the dried biomass before step a). 5. The method of claim 4, further comprising passing the milled dry biomass through a mesh, optionally a 50-200 [mu]m mesh or a 100 [mu]m mesh. The method according to any one of claims 1 to 5, further comprising the step of degreasing the biomass before step a). 7. The method of any one of claims 1-6, wherein the mild alkaline conditions include a pH of greater than 7 and less than or equal to about 11, a pH of about 9 to about 11, or a pH of about 10. 8. The method of any one of claims 1-7, wherein step a) is carried out at a temperature of about 90<0>C to about 100<0>C. The method of claim 8, wherein step a) is performed at a temperature of about 90°C to about 95°C. 10. The method of any one of claims 1-9, wherein step a) is performed for a period of about 15 minutes to about 2 hours, about 30 to about 90 minutes, or about 1 hour. 11. The method of any one of claims 1-10, wherein step b) is performed at a pH of about 7 to about 11. The method according to any one of claims 1 to 11, wherein step b) is carried out at a temperature of about 50°C to about 80°C. 13. The method of claim 12, wherein step b) is performed at a temperature of about 55°C. 14. The method of any one of claims 1-13, wherein step b) is performed for a time of about 15 minutes to about 2 hours, about 30 to about 90 minutes, or about 1 hour. 15. The method of any one of claims 1-14, wherein the amount of biomass in the aqueous solution is about 0.5% to about 20% (w/v). 16. The method of any one of claims 1-15, wherein the proteolytic enzyme comprises subtilisin. 17. The method of claim 16, wherein the subtilisin is from Bacillus licheniformis. 18. The method of any one of claims 1-17, wherein step c) comprises centrifuging the proteolytic enzyme treated slurry of b) to obtain the liquid and solid fractions. 19. The method of any one of claims 1-18, further comprising inactivating the proteolytic enzyme after step b). 20. The method of claim 19, wherein the quenching step is heat quenching. 21. The method of claim 20, wherein the heat inactivation is performed at a temperature of about 80<0>C to about 100<0>C for a time of about 5 to about 30 minutes. (I) b) treating the proteolytic enzyme treatment slurry with a solution containing divalent cations, and/or (ii) treating the liquid fraction of c) with a solution containing divalent cations. 22. The method of any one of claims 1-21, further comprising: Said _solution containing CaCl _2, MgCl _2, MnCl ₂ and at least one of FeCl _2, The method of claim 22. 24. The step of c) comprising treating the liquid fraction with a solution containing divalent cations to precipitate phytate, further comprising the step of isolating the liquid fraction from the phytate precipitate. The method described in. 25. The method of any one of claims 22-24, wherein the solution containing divalent cations is used in an amount of about 1.5 to about 20 x equivalents. 26. The method of any one of claims 1-25, further comprising subjecting the liquid fraction to size exclusion chromatography or filtration. 27. The method of any one of claims 1-26, further comprising concentrating the liquid fraction. 28. The method of any one of claims 1-27, wherein the biomass is cereal biomass, plant biomass, distillers dried grains (DDG), soybean biomass, rapeseed meal or flaxseed meal. 29. The method of claim 28, wherein the biomass is soybean biomass. 30. The method of claim 29, wherein the soybean biomass is okara. 31. The method according to any one of claims 1 to 30, further comprising drying the liquid fraction to obtain a protein and/or peptide rich dried product. 32. The method of claim 31, wherein the protein and/or peptide rich dry product has at least 50% less residual trypsin inhibitor activity than commercial soy protein concentrate (SPC). 33. The method of claim 31 or 32, wherein the protein and/or peptide rich dry product has a residual phytate content that is at least 60% lower than commercial soy protein concentrate (SPC). 34. The method of claim 32 or 33, wherein the commercial SPC is Archon(R) F. 35. The method of any one of claims 1-34, further comprising drying the solid fraction to obtain a fiber-rich dry product. 36. The method of claim 35, wherein the fiber-rich dry product has a carbohydrate content of greater than or equal to about 70% and a protein content of less than or equal to about 10%. The following features:
a) greater than 80% water solubility over a pH of about 3 to about 11;
b) a protein and/or peptide content of about 40% or higher;
c) at least 75% of the proteins and/or peptides in the extract have a molecular weight of less than 20 kDa;
d) A protein and/or peptide rich dry biomass extract with reduced trypsin inhibitory activity and phytate content compared to commercial soy protein concentrate (SPC). 38. Dry protein extract enriched in proteins and/or peptides according to claim 37, having a carbohydrate content of about 20% carbohydrate and/or a lipid content of about 10%. A dry biomass extract rich in protein and/or peptide according to claim 37 or 38, obtained by a method according to any one of claims 31 to 33. A fiber-rich dry biomass extract obtained by the method of claim 35 or 36. Beverage, cosmetic, comprising the protein and/or peptide rich dry biomass extract of any one of claims 37 to 39 and/or the fiber rich dry biomass extract of claim 40. Food or feed products. A method for producing a beverage, cosmetic, food or feed product, comprising: (i) carrying out the method according to any one of claims 31 to 33 to obtain a dry product rich in proteins and/or peptides. And (ii) incorporating said protein and/or peptide rich dry product into a beverage, cosmetic, food or feed composition. A method of producing a food or feed product, the method comprising: (i) performing the method of claim 35 or 36 to obtain a fiber-rich dry product; and (ii) the fiber-rich dry product. Incorporating into a food or feed composition.